Legionella pneumophila is a facultative intracellular pathogen that grows inside protozoans in fresh water sources. Susceptible humans may develop a fulminant pneumonia called Legionnaires disease following inhalation of contaminated water aerosols. This opportunistic pathogen contributes significantly to community-acquired and nosocomial pneumonia infection rated and is associated with serious morbidity and mortality. During human infection, and in animal models, virulent L. pneumophila elicits a cellular immune response, including recruitment and activation of macrophages, which serve as the primary cellular target for entry and subsequent growth and the production of specific cytokines. A series of prior studies has demonstrated that the outcome of the interaction with macrophages depends upon the expression of L. pneumophila genes, some of which may be regulated by intracellular environmental signals. Only a few genes that apparently are involved in this interaction have been discovered, and the definitive role of any of these in the pathogenesis of infection and disease in unknown. Studies on a L. pneumophila mutant that lacks the capacity to produce protease strongly suggest that virulence was attenuated in an animal model, and furthermore, that this effect might be directly related to impairment of the host s immune response, including cytokine production. Other studies demonstrated that prior growth of laboratory passaged strains in Acanthamoeba enhanced the invasiveness of L. pneumophila into macrophages. This was associated with the expression of new polypeptides. The specific aims of the proposal are to: 1) further define the contribution of the protease protein to L. pneumophila pathogenesis and host immune response: and 2) detect and define the role of new genes, not yet discovered, that are involved in intracellular growth in eukaryotic cells. Dr. Tompkins propose to study the cell biology of he interaction of macrophages with wild-type strains and mutants to characterize and define the steps in intracellular growth governed by each gene product. She will also use two new genetic selection methods to isolate and identify new genes involved in intracellular growth. Thus, the proposal incorporates novel molecular and genetic methods and utilizes contemporary microscopy techniques, including laser confocal microscopy, to further define and characterize the cell biology of Legionella in eukaryotic cells.